Peptide-major histocompatibility complex protein complexes (pMHCs) on antigen-presenting cells (APCs) are central to T cell activation. Within minutes of peptide-specific T cells interacting with ...APCs, pMHCs on APCs formed clusters at the site of T cell contact. Thereafter, these clusters were acquired by T cells and internalized through T cell receptor-mediated endocytosis. During this process, T cells became sensitive to peptide-specific lysis by neighboring T cells (fratricide). This form of immunoregulation could explain the "exhaustion" of T cell responses that is induced by high viral loads and may serve to down-regulate immune responses.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by ...activation of phospholipase C-γ1 (PLC-γ1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-γ1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-γ1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Activation of naive T cells generally requires T cell receptormediated contact with MHC-bound peptides on viable antigenpresenting cells such as dendritic cells (DC). Here evidence is presented that ...dissociated cell membrane fragments from a DC line can be used as an effective substitute for viable DC. Ultracentrifuged material derived from sonicates of IFN-γ-matured DC is enriched in small membrane vesicles that closely resemble exosomes. When complexed with MHC class I-restricted specific peptide, vesicles from DC sonicates generate strong responses by purified naive CD8⁺ cells in vitro in the absence of normal antigenpresenting cells and can also efficiently prime T cells for tumor rejection in vivo. Both in terms of total yields from DC and relative immunogenicity, membrane vesicles from DC sonicates are much more effective than classic exosomes and may be a valuable tool for tumor immunotherapy.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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The concurrent implementation of a proteome-wide serine hydrolase selectivity screen with traditional efforts to optimize fatty acid amide hydrolase (FAAH) inhibition potency led to ...the expedited discovery of a new class of exceptionally potent (
K
i
<
300
pM) and unusually selective (>100-fold selective) inhibitors. The iterative inhibitor design and evaluation with assistance of the selectivity screen served to differentiate otherwise indistinguishable inhibitors permitting the simultaneous optimization of potency and selectivity. Significantly, the simultaneous assessment of all potential competitive enzymes with the selectivity screen does not require the use of expressed or purified enzymes or a competitive substrate, no modification of the inhibitors is required, and the relative potency for competitive enzymes can be quantified (IC
50’s) including those that lack known substrates or function.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major ...histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-gamma1 and entry of extracellular Ca(2+) following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Key derivatives and analogues of fostriecin were prepared and examined that revealed a fundamental role for the unsaturated lactone and confirmed the essential nature of the phosphate monoester. ...Thus, an identical 200-fold reduction in protein phosphatase 2A (PP2A) inhibition is observed with either the saturated lactone (7) or with an analogue that lacks the entire lactone (15). This 200-fold increase in PP2A inhibition attributable to the unsaturated lactone potentially may be due to reversible C269 alkylation within the PP β12−β13 active site loop accounting for PP2A/4 potency and selectivity.
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IJS, KILJ, NUK, PNG, UL, UM
Anti-allergic effects of the hot water extract of
Rosae multiflorae fructus
(Rosae extract), which has long been used in oriental medicine for treatment of various diseases, were explored with a ...chicken ovalbumin (cOVA)-induced mouse model of food allergy. Compared to the sham mice to show severe allergic symptoms (i.e., anaphylaxis, diarrhea and decrease in the body temperature) following oral cOVA challenge, the Rosae extract-treated mice showed a marked improvement in those symptoms. Histology data demonstrated that Rosae extract treatment resulted in a amelioration in the intestinal inflammatory lesion and a reduction in the numbers of mast cells and eosinophils in the small intestine. Studies using DO11.10 TCR transgenic T cells indicated that Rosae extract had an activity to subdue the antigen-specific T cell activation/proliferation in vivo and thereby to lower the level of Th2 cytokine production by T cells during the antigen-specific immune response. Moreover, passive systemic anaphylaxis study showed that the extract also had an activity to inhibit the mast cells function in vivo, i.e., release of granules triggered by specific IgE-antigen interaction. Altogether, the results from this study not only imply a potential clinical application of Rosae extract in prevention and treatment of food allergy but also clearly elucidate the immunoregulatory mechanisms of Rosae extract underlying its anti-allergic effect.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The total synthesis and evaluation of three key ramoplanin aglycon analogues are detailed. The first (5a) represents replacement of the labile depsipeptide ester with a stable amide (HAsn2 → Dap2) ...with removal of the HAsn pendant carboxamide, and it was found to be slightly more potent than the natural aglycon in antimicrobial assays providing a new lead structure with an improved profile and a more stable and accessible macrocyclic template on which to conduct structure−function studies. In contrast, a second amide analogue 5b which contains a single additional methylene relative to 5a (HAsn2 → Dab2) was found to be inactive in antimicrobial assays (>100-fold loss in activity). The third key analogue 5c in which the Asn lipid side chain was replaced with an acetyl group revealed that it contributes significantly to the antimicrobial activity (16-fold) of the ramoplanins, but is not essential.
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IJS, KILJ, NUK, PNG, UL, UM
Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of ...interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.
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